Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan, which is present in a thick layer in gram-positive bacteria. In a Gram stain test, gram-positive bacteria retain the crystal violet dye, while a counterstain (commonly safranin or fuchsine) added after the crystal violet gives all gram-negative bacteria a red or pink coloring.
Among the gram-positive bacteria are the genera Clostridium, Listeria, Bacillus, Staphylococcus and Streptococcus, which are all pathogenic in humans. The genus Clostridium contains around 100 species that include common free-living bacteria, as well as important pathogens.
One of the Clostridium species, Clostridium perfringens, is a ubiquitous spore-forming pathogen that produces at least 15 different potent toxins that are responsible for severe diseases in humans and animals (Popoff and Bouvet, 2009).
C. perfringens causes a wide range of symptoms in humans, from food poisoning to gas gangrene, which is necrosis, putrefaction of tissues, and gas production. The gases form bubbles in muscle (crepitus) and the characteristic smell in decomposing tissue. After rapid and destructive local spread (which can take only hours), systemic spread of bacteria and bacterial toxins may cause death. This is a problem in major trauma and in military contexts.
It is generally accepted that C. perfringens toxinotype A is the cause of several different diseases in broiler chickens, including clinical necrotic enteritis, which is characterized by outbreaks of spiking mortality. This classical acute clinical form of necrotic enteritis has emerged in broilers in the European Union following the ban on antimicrobial growth promoters in animal feed in 2006 (Van Immerseel et al., 2009). Many animals die without premonitory signs, and mortality can, in some cases, rise up to 50% (Wijewanta and Senevirtna, 1971).
Clostridium perfringens, however, also can cause an unapparent infection or a subclinical disease characterized by the presence of small ulcerative lesions in the mucosa of the small intestine. It is generally accepted that these small intestinal lesions lead to poor digestion and absorption. Moreover, multifocal liver lesions can be found during meat inspection at slaughter. These red or white foci are due to colonization of the liver by high numbers of C. perfringens, resulting in multifocal cholangiohepatitis (Kaldhusdahl and Hofshagen, 1992).
All of these lesions result in reduced weight gain and increased feed conversion ratio (Kaldhusdahl et al., 2001). Feed conversion ratio is defined as the ratio of feed mass input to body mass output. The economic impact of subclinical necrotic enteritis is considered to be much more important than that of clinical necrotic enteritis. It has been suggested that subclinical necrotic enteritis would occur in 20% of the broilers. This results in an increase in feed conversion rate of 10.9%, having a severe economic impact.
Worldwide, necrotic enteritis is probably one of the most widespread enteric diseases in poultry, with considerable consequences for the performance of affected flocks. It can present as a sudden increase in mortality or as an insidious subclinical disease. The indication of necrotic enteritis is the presence of necrotic lesions in the small intestinal mucosa. The diagnosis of necrotic enteritis is made at necropsy.
Besides causing both severe health issues among affected animals and great economical losses, pathogens like Clostridium perfringens can also be spread from infected animals to humans. People handling these animals or consumers of the animals or animal products risk getting infected and, as described above, develop serious illness.
The emerging problem with antimicrobial resistance resulted in the ban of growth promoting antibiotics in the European Union in 2006. This calls for new, alternative methods to counteract pathogenic bacteria in both humans and animals.
It is well known in the art that various short chain fatty acids as well as their respective glycerol monoesters have antibacterial properties. There are several benefits associated with distributing the short chain fatty acids as glycerol esters. Glycerol esters are less corrosive than the corresponding free acids, facilitating handling and transportation of the products. Some of the short chain fatty acids, like for example butyric acid and valeric acid, have a very unpleasant smell. The corresponding glycerol esters of these acids are more or less odorless. Additionally, glycerol esters bind the short chain fatty acids, enabling them to reach further down in the gastrointestinal tract of a human or an animal before being adsorbed to the bloodstream.
Quite a lot of studies have been published on the use of glycerol esters of propionic acid and butyric acid as antibacterial agents. However, the specific antibacterial action of glyceryl monovalerate has not been described.
The present invention shows that glycerol monovalerate is much more effective than either glyceryl monopropionate or glyceryl monobutyrate for reducing and/or inhibiting the gram-positive bacterium Clostridium perfringens in vitro. The antibacterial action of glyceryl monovalerate against Clostridium perfringens is confirmed in an in vivo study on broiler chicken.